CN111411056A - Lactobacillus paracasei subspecies tenacious strain, screening method and application - Google Patents
Lactobacillus paracasei subspecies tenacious strain, screening method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a Lactobacillus paracasei subspecies tenacious strain, a screening method and application thereof, wherein the screening method of the Lactobacillus paracasei subspecies tenacious strain comprises the following steps: placing the lactobacillus to be screened into a culture dish containing a primary screening culture medium for constant-temperature culture, inoculating the lactobacillus obtained after primary screening into fresh milk for fermentation culture, and obtaining a fermentation composition; carrying out bacteriostatic activity detection; and respectively placing the single colonies which pass the activity detection in a primary screening culture medium and an anaerobic culture medium for continuous culture to obtain the lactobacillus paracasei tough subspecies FX-6-1 strain with good antibacterial activity. The screening method of the lactobacillus paracasei subspecies tough strain is simple and feasible, has low operation cost, can obtain broad-spectrum and efficient novel antibacterial substances, promotes the development and application of the novel antibacterial substances, and has wide market.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus paracasei subspecies strain, a screening method and application thereof.
Background
At present, in the processes of processing, storage, transportation and consumption, the food spoilage is caused by a plurality of reasons, including physical factors, chemical factors and biological factors, wherein the food spoilage caused by microbial contamination is the most important and common. The food is rotten and deteriorated due to the breeding of microorganisms, and meanwhile, serious harm is brought to the food safety. Although the traditional heat sterilization can effectively inhibit the pollution of microorganisms, the nutrition, the texture and the color and flavor types of the food are damaged to different degrees while the sterilization is carried out, and simultaneously, a large amount of energy consumption is generated. In modern food industry, food preservatives are widely used to extend the shelf life of food, and have become an indispensable food additive in modern food production. In modern food industry and planting and breeding industry, the irregular use and residual hidden danger of chemical preservatives, pesticides and antibiotics are attracting more and more attention of consumers, and people tend to consume food resources from natural sources.
Lactobacillus paracasei (L actinobacillus paracasei), facultative anaerobic, immotile, spore-free bacillus or long-rod bacteria, gram positive, catalase negative, Lactobacillus paracasei widely exist in traditional fermented dairy products and human gastrointestinal tracts, have physiological functions of regulating intestinal flora balance, enhancing immune function, preventing diseases and the like, and the screening method of the existing Lactobacillus paracasei is not reported yet.
Therefore, in order to solve the problems of chemical preservatives in the food industry and the demand of broad-spectrum efficient natural preservative and fresh-keeping agents, a method for separating and screening lactobacillus paracasei strains with antibacterial activity from bacterial liquid to be screened is urgently needed.
Through the above analysis, the problems and defects of the prior art are as follows: the existing screening method of lactobacillus paracasei is not reported yet; meanwhile, the problem of chemical preservatives widely exists in the food industry, and broad-spectrum efficient natural preservative is urgently needed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a lactobacillus paracasei subspecies strain, a screening method and application thereof.
The invention is realized by the method for screening the lactobacillus paracasei subspecies tenacious strain, which comprises the following steps:
step one, primary screening: putting the selected strains of the lactobacillus to be screened into a culture dish containing a primary screening culture medium, and putting the culture dish into a constant-temperature shaking table at the temperature of 30-35 ℃ for culturing for 4-5 days;
step two, inoculating and culturing: inoculating lactobacillus obtained after primary screening culture into fresh milk, and placing the fresh milk in a biochemical incubator at the temperature of 30-35 ℃ for fermentation culture for 3-4 days to obtain a fermentation composition;
step three, activity detection: selecting and streaking the fermented composition, taking escherichia coli as an indicator bacterium, and carrying out bacteriostatic activity test detection on the obtained single colony;
placing the single bacterial colony passing the activity detection into a primary screening culture medium, and performing shake culture for 8-10 days; 2ml of the cultured bacterial liquid is put into an anaerobic bottle with an anaerobic culture medium for culturing for 25-30 days;
and step five, placing the anaerobic bottle into an anaerobic culture tank at the temperature of 30 ℃ for culturing for 20 days, and continuously filling nitrogen in the culture process to obtain the lactobacillus paracasei strain FX-6-1 with good antibacterial activity.
Further, in the first step, the preparation method of the primary screening culture medium comprises the following steps:
(1) mixing Na in parts by mass2S2O310 to 15 parts of KNO38 to 12 parts of (NH)4)2HPO45-7 parts of cysteine and 1-2 parts of cysteine are mixed to obtain a mixed solution;
(2) adding 5% alkaline re-reddish ethanol solution into the mixed solution, and adjusting the pH value to 7.2-7.4;
(3) setting the heating temperature to be 120-125 ℃, and sterilizing for 15-20 min;
(4) and cooling at room temperature, adding 15-20 parts of vitamin nutrient solution, and uniformly stirring to obtain a primary screening culture medium.
Furthermore, thiamine in the vitamin nutrient solution is 0.12-0.15 mu g/L, pyridoxine is 0.13-0.16 mu g/L, calcium pantothenate is 0.15-0.2 mu g/L, zinc sulfate is 3-5 mg/L, and nicotinic acid is 0.11-0.13 mu g/L.
Further, in the first step, the rotating speed of the constant-temperature shaking table is 180-190 rpm/min.
Further, in the third step, the step of performing a bacteriostatic activity test on the obtained single colony comprises:
(1) preparing 25ml of culture medium by using a phi 90 plate, wherein the addition amount of agar is 0.75%;
(2) sterilizing the culture medium at 150 ℃ for 5min at 120-;
(3) adding Escherichia coli, mixing, and pouring into flat plate;
(4) after the culture medium is solidified, punching by using a 6mm puncher, and respectively adding the standard sample and the detected sample into different holes;
(5) and after 3-8 hours of low-temperature diffusion, culturing in an upright incubator, and calculating the bacteriostatic activity according to the diameter of a bacteriostatic zone.
Further, in the fourth step, the temperature of the shaking table culture is 30-35 ℃, and the rotation speed of the shaking table is 180-185 r/min.
Further, in the fourth step, the anaerobic culture medium is composed of, by mass, 15-17 parts of MnSO410 to 12 portions of Na2S2O35-8 parts of KH2HPO43 to 5 parts of NH4Cl, 1.5-2 parts of CaCl2And 0.5-1 part of FeCl3Mixing, boiling, cooling, adjusting pH to neutral, adding 1-1.2 ml indicator, and charging N2Bottling, and sterilizing at 120-125 deg.C for 20-25 min.
Further, the indicator is an anaerobic indicator of methylene blue.
The invention also aims to provide a tough subspecies strain of lactobacillus paracasei obtained by screening by using the screening method of the tough subspecies strain of lactobacillus paracasei, wherein the tough subspecies strain of lactobacillus paracasei FX-6-1 is preserved in China center for type culture Collection at Wuhan university, Wuhan City, 2014 at 23 months 02, and the preservation number is CCTCC M2014043.
The invention also aims to provide the application of the lactobacillus paracasei subspecies tenacious strain in preparing antibacterial medicaments.
By combining all the technical schemes, the invention has the advantages and positive effects that: the screening method of the lactobacillus paracasei subspecies tough strain is simple and feasible, has low operation cost, can obtain broad-spectrum and efficient novel antibacterial substances, promotes the development and application of the novel antibacterial substances, and has wide market.
The antibacterial composition produced by fermenting the lactobacillus paracasei tough subspecies strain obtained by screening has stable property and wide antibacterial spectrum, can inhibit gram-positive bacteria and gram-negative bacteria in bacteria, and has an inhibiting effect on partial mold.
Drawings
FIG. 1 is a flow chart of a method for screening a Lactobacillus paracasei subspecies tenacious strain according to an embodiment of the present invention.
FIG. 2 is a flow chart of a method for preparing a prescreening medium according to an embodiment of the invention.
FIG. 3 is a flowchart of the bacteriostatic activity test for the single colony obtained in the embodiment of the present invention.
FIGS. 4 to 5 are schematic diagrams showing the colony morphology of Lactobacillus paracasei subspecies FX-6-1 obtained by screening and separation according to the embodiment of the present invention.
FIGS. 6 to 7 are schematic diagrams showing morphological characteristics of Lactobacillus paracasei subspecies tenacious strain observed under a microscope oil microscope.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a Lactobacillus paracasei subspecies tenacious strain, a screening method and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
As shown in FIG. 1, the screening method of Lactobacillus paracasei subspecies tenacious strain provided by the embodiment of the present invention comprises the following steps:
s101, primary screening: putting the selected strains of the lactobacillus to be screened into a culture dish containing a primary screening culture medium, and putting the culture dish into a constant-temperature shaking table at the temperature of 30-35 ℃ for culturing for 4-5 days;
s102, inoculating and culturing: inoculating lactobacillus obtained after primary screening culture into fresh milk, and placing the fresh milk in a biochemical incubator at the temperature of 30-35 ℃ for fermentation culture for 3-4 days to obtain a fermentation composition;
s103, activity detection: selecting and streaking the fermented composition, taking escherichia coli as an indicator bacterium, and carrying out bacteriostatic activity test detection on the obtained single colony;
s104, placing the single bacterial colony passing the activity detection into a primary screening culture medium, and performing shake culture for 8-10 days; 2ml of the cultured bacterial liquid is put into an anaerobic bottle with an anaerobic culture medium for culturing for 25-30 days;
s105, placing the anaerobic bottle into an anaerobic culture tank at the temperature of 30 ℃ for culturing for 20 days, and continuously filling nitrogen in the culture process to obtain the Lactobacillus paracasei tough subspecies FX-6-1 strain with good antibacterial activity.
As shown in fig. 2, the preparation method of the primary screening medium provided by the embodiment of the present invention comprises:
s201, mixing Na in parts by mass2S2O310 to 15 parts of KNO38 to 12 parts of (NH)4)2HPO45-7 parts of cysteine and 1-2 parts of cysteine are mixed to obtain a mixed solution;
s202, adding a 5% alkaline re-reddish ethanol solution into the mixed solution, and adjusting the pH value to 7.2-7.4;
s203, setting the heating temperature to be 120-125 ℃, and sterilizing for 15-20 min;
and S204, cooling at room temperature, adding 15-20 parts of vitamin nutrient solution, and uniformly stirring to obtain a primary screening culture medium.
As shown in fig. 3, the detection of the bacteriostatic activity test on the obtained single colony provided by the embodiment of the present invention includes:
s301, preparing 25ml of culture medium by using a phi 90 plate, wherein the addition amount of agar is 0.75%;
s302, sterilizing the culture medium at the temperature of 150 ℃ for 5min at 120-;
s303, adding escherichia coli, uniformly mixing, and pouring into a flat plate;
s304, after the culture medium is solidified, punching by using a 6mm puncher, and respectively adding the standard sample and the detected sample into different holes;
s305, after low-temperature diffusion for 3-8 hours, placing the mixture in an incubator for culture, and calculating the bacteriostatic activity according to the diameter of a bacteriostatic zone.
The lactobacillus paracasei subspecies strain FX-6-1 provided by the embodiment of the invention is preserved in 23 days 02 and 2014 in the china type culture collection located in the university of wuhan in wuhan, with the preservation number of CCTCC M2014043.
The present invention will be further described with reference to the following examples.
Example 1: screening and isolation of Lactobacillus paracasei tough subspecies FX-6-1 Strain
Inoculating the selected strain of lactobacillus to be screened into pure milk, performing static fermentation culture at 30 ℃ for 24h, mixing fermented curds uniformly, and diluting the liquid fermented milk to 10% with 0.9% sterile normal saline-6～10-8And after vortex mixing, sucking 0.1m L diluted samples to the surfaces of an MC solid culture medium and an MRS solid culture plate, uniformly coating, carrying out sealed culture at 30 ℃ for 24-48 h until a single bacterial colony grows out, picking the single bacterial colony generating a clear calcium ring, purifying through the MRS agar plate again to obtain single bacterial colony strains, and collecting 50 lactic acid bacteria in total.
Inoculating the separated acid-producing single colony strain into MRS liquid culture medium, and statically sealing at 30 ℃ for overnight culture. And then adding the activated lactobacillus bacterial liquid into pure milk according to the inoculation amount of 5% (v/v), uniformly mixing, performing static seal culture at 30 ℃ for 24 hours, centrifuging the fermentation liquid (4,000r/min, 20min), passing through a membrane (0.45 mu m filter membrane), and performing freeze drying to obtain a freeze-dried sample.
Escherichia coli is used as an indicator bacterium, and an Oxford cup agar diffusion method is adopted to screen a Lactobacillus paracasei tough subsp FX-6-1 (L actinobacillus paracasei subsp. tolerans FX-6-1) strain with the best antibacterial activity.
The lactobacillus paracasei tough subspecies FX-6-1 strain has been preserved in 2014 23.02 in the chinese type culture collection, with the preservation number of CCTCC M2014043, located at the university of wuhan, wuhan.
Example 2: gram staining of Lactobacillus paracasei tough subspecies FX-6-1 Strain
The bacterial colony of Lactobacillus paracasei tough subspecies FX-6-1 cultured on a one-ring MRS plate culture medium is picked by using an inoculating ring, gram staining is carried out after smearing, and the morphological characteristics of the lactobacillus strain are observed by an oil mirror. The bacterial strains are positive through gram staining and are short rod-shaped, the bacterial strains are adhered into a short chain arrangement, and the bacterial strains are free of flagella and spore formation and are typical characteristics of the lactobacillus (see figures 6-7).
Example 3: determination of bacteriostatic Activity
Sucking a bacteriostatic composition solution generated by fermenting 2m L Lactobacillus paracasei subspecies FX-6-1 strain to an Oxford cup, statically culturing the bacteria at a constant temperature of 37 ℃ for 18h (culturing the mold at 30 ℃ for 24h by adopting a PDA solid culture medium), and measuring the diameter of a bacteriostatic zone by using a vernier caliper.
The experimental results are shown in Table 1, and it can be seen that the bacteriostatic composition (1g/m L) produced by fermenting the Lactobacillus paracasei tough subspecies FX-6-1 strain has a wide antibacterial spectrum, has a significant inhibitory effect on part of gram-positive bacteria and gram-negative bacteria of bacterial strains, and also has inhibitory effects on Aspergillus flavus, Aspergillus niger, Rhizopus nigrosins and Penicillium in the mold to different degrees.
TABLE 1 results of the measurement of bacteriostatic Activity
| Indicator bacterium | Bacteriostatic activity |
| Escherichia coli (E.coli) | +++ |
| Staphylococcus aureus (S.aureus) | +++ |
| Bacillus thuringiensis (B.thuringiensis) | +++ |
| Salmonella (S.enterica) | +++ |
| Dysentery bacillus (S.dysenteriae) | +++ |
| Aspergillus flavus (A. flavus) | + |
| Aspergillus niger (A.niger) | +++ |
| Rhizopus nigricans (R. nigricans) | ++ |
| Penicillium glaucum (Pen. glaucum) | ++ |
Note: +: the diameter of the bacteriostatic circle is 5-10 mm; ++: the diameter of the bacteriostatic circle is 10-20 mm; +++: the diameter of the bacteriostatic zone is more than 20 mm.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for screening a Lactobacillus paracasei subspecies tenacious strain, which is characterized by comprising the following steps:
step one, primary screening: putting the selected strains of the lactobacillus to be screened into a culture dish containing a primary screening culture medium, and putting the culture dish into a constant-temperature shaking table at the temperature of 30-35 ℃ for culturing for 4-5 days;
step two, inoculating and culturing: inoculating lactobacillus obtained after primary screening culture into fresh milk, and placing the fresh milk in a biochemical incubator at the temperature of 30-35 ℃ for fermentation culture for 3-4 days to obtain a fermentation composition;
step three, activity detection: selecting and streaking the fermented composition, taking escherichia coli as an indicator bacterium, and carrying out bacteriostatic activity test detection on the obtained single colony;
placing the single bacterial colony passing the activity detection into a primary screening culture medium, and performing shake culture for 8-10 days; 2ml of the cultured bacterial liquid is put into an anaerobic bottle with an anaerobic culture medium for culturing for 25-30 days;
and step five, placing the anaerobic bottle into an anaerobic culture tank at the temperature of 30 ℃ for culturing for 20 days, and continuously filling nitrogen in the culture process to obtain the lactobacillus paracasei strain FX-6-1 with good antibacterial activity.
2. The method for screening Lactobacillus paracasei subspecies tenacious strain according to claim 1, wherein in step one, the primary screening medium is prepared by:
(1) mixing Na in parts by mass2S2O310 to 15 parts of KNO38 to 12 parts of (NH)4)2HPO45-7 parts of cysteine and 1-2 parts of cysteine are mixed to obtain a mixed solution;
(2) adding 5% alkaline re-reddish ethanol solution into the mixed solution, and adjusting the pH value to 7.2-7.4;
(3) setting the heating temperature to be 120-125 ℃, and sterilizing for 15-20 min;
(4) and cooling at room temperature, adding 15-20 parts of vitamin nutrient solution, and uniformly stirring to obtain a primary screening culture medium.
3. The method for screening Lactobacillus paracasei subspecies tenacious strain according to claim 2, wherein the vitamin nutrient solution contains thiamine in an amount of 0.12 to 0.15 μ g/L, pyridoxine in an amount of 0.13 to 0.16 μ g/L, calcium pantothenate in an amount of 0.15 to 0.2 μ g/L, zinc sulfate in an amount of 3 to 5 mg/L, and nicotinic acid in an amount of 0.11 to 0.13 μ g/L.
4. The method for screening Lactobacillus paracasei subspecies tenacious strain according to claim 1, wherein in the first step, the rotation speed of the constant temperature shaker is 180 to 190 rpm/min.
5. The method for screening the Lactobacillus paracasei subspecies tenacious strain according to claim 1, wherein the step three, the test for the bacteriostatic activity of the obtained single colony comprises:
(1) preparing 25ml of culture medium by using a phi 90 plate, wherein the addition amount of agar is 0.75%;
(2) sterilizing the culture medium at 150 ℃ for 5min at 120-;
(3) adding Escherichia coli, mixing, and pouring into flat plate;
(4) after the culture medium is solidified, punching by using a 6mm puncher, and respectively adding the standard sample and the detected sample into different holes;
(5) and after 3-8 hours of low-temperature diffusion, culturing in an upright incubator, and calculating the bacteriostatic activity according to the diameter of a bacteriostatic zone.
6. The method for screening Lactobacillus paracasei subspecies tenacious strain according to claim 1, wherein in step four, the temperature of the shaking table culture is 30 to 35 ℃ and the shaking table rotation speed is 180 to 185 rpm.
7. The screening method of Lactobacillus paracasei subspecies tenacious strain according to claim 1, wherein in step four, the anaerobic culture medium is composed of 15-17 parts of MnSO by weight410 to 12 portions of Na2S2O35-8 parts of KH2HPO43 to 5 parts of NH4Cl, 1.5-2 parts of CaCl2And 0.5-1 part of FeCl3Mixing, boiling, cooling, adjusting pH to neutral, adding 1-1.2 ml indicator, and charging N2Bottling, and sterilizing at 120-125 deg.C for 20-25 min.
8. The method of screening a Lactobacillus paracasei subspecies tenacious strain according to claim 7, wherein the indicator is an anaerobic indicator of methylene blue.
9. The Lactobacillus paracasei subspecies strain screened by the screening method of the Lactobacillus paracasei subspecies strain according to any one of claims 1 to 8, wherein the Lactobacillus paracasei subspecies strain FX-6-1 is preserved in the China center for type culture Collection, CCTCC M2014043, located in Wuhan university, Wuhan, 2014, 23 months.
10. Use of a strain of Lactobacillus paracasei subspecies tenacious according to claim 9 for the preparation of an antibacterial medicament.
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| CN114480214A (en) * | 2022-03-07 | 2022-05-13 | 西藏安琪生物科技有限公司 | Lactobacillus paracasei separated from Tibet Aliyak milk keloid and application thereof |
| CN116640700A (en) * | 2023-06-24 | 2023-08-25 | 西南大学 | Lactobacillus paracasei F50 for inhibiting aspergillus flavus growth and toxin production and application thereof |
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